Development and Characterization of a Dynamic Triculture Blood-Brain Barrier Model

Many in vitro blood-brain barrier (BBB) models lack physiological flow and key neurovascular interactions, limiting their ability to reproduce BBB properties in vivo. In this study, we investigated whether incorporation of primary human astrocytes and pericytes together with flow conditions could improve BBB properties in hCMEC/D3-based models.

Static and dynamic BBB models were established using hCMEC/D3 endothelial cells cultured alone or in triculture with primary human astrocytes and pericytes. Barrier integrity was assessed by transendothelial electrical resistance (TEER) measurements, permeability of Lucifer Yellow (LY), doxorubicin (DOX), and mitoxantrone (MIT), and expression analysis of BBB-associated genes.

Astrocyte/pericyte co-culture improved BBB properties compared with endothelial monocultures, resulting in higher TEER values and lower permeability of LY, DOX, and MIT. The dynamic culture, where BBB was exposed to a millifluidic flux mimicking blood flow, further enhanced barrier competency compared with corresponding static conditions, with the lowest permeability consistently observed in the dynamic triculture model. Compared with static triculture conditions, LY, DOX, and MIT permeability in the dynamic triculture model were reduced by approximately 20%, 15%, and 30%, respectively. Compared with static endothelial monocultures, the dynamic triculture model reduced LY, DOX, and MIT permeability by 30%, 35%, and 60%, respectively. Reduced permeability was accompanied by increased CLDN5 and ABCG2/BCRP expression (50% and 60%, respectively), suggesting progression toward a more mature BBB phenotype.

In conclusion, the combination of astrocyte/pericyte co-culture and physiological flow generated the most restrictive BBB phenotype among the tested conditions. As a step toward reproducing the complex blood-brain and blood-tumor interactions occurring in vivo, this platform may represent an innovative and reliable tool for future neurobiology and drug permeability studies.

Acknowledgements: This project has received funding from the European Union's Horizon Europe research and innovation programme under the Marie Skłodowska-Curie Postdoctoral Fellowship grant agreement No. 101151942 (GBM4EPI).